(Note: This application references a number of different publications as indicated throughout the specification by one or more reference numbers in brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below in the section entitled “References.” Each of these publications is incorporated by reference herein.)
Conventional high-resolution microscopes capable of imaging bacteria have limited depth-of-field and typically require complex objective lenses with tight alignment tolerances. As a result, direct observation of prokaryotes (bacteria and archaea) in their native environments has yet to be performed in most parts of the Arctic and Antarctic, around hydrothermal vents, and in the majority of the open ocean [1]. Quantifying prokaryotic behavior in situ is important for understanding large-scale marine processes such as carbon cycling [2, 3]. It is also of great interest for the investigation of the possibility of life in extraterrestrial Ocean Worlds, such as Enceladus and Europa [4], but instruments for unambiguous detection of prokaryotes in planetary environments don't yet exist. Some microscopes have flown in space, but as yet no microscopic observations have been made on Mars with the resolution required to detect bacteria [5, 6]. For all such remote deployment scenarios, a compact, robust microscope capable of operating in an ambient environment is required.
Digital holographic microscopy [7] has a number of advantages over conventional microscopy for remote autonomous deployment, including robustness (no moving parts, as no focus mechanism is needed), high throughput, and compressed sensing (i.e., the entire 3-d sample volume is encoded in each 2-d frame capture). As a result, this technique is beginning to see application beyond the laboratory [8,9]. Our previous “common path” digital holographic microscope [DHM] design [10] reported microbial imaging in Greenland sea ice [11], but its reliance on classical lens-based optics left this prototype larger than desirable for robotic deployment. On the other hand, lensless holography, with no imaging optics between the light source and detector [12-16], can enable compact, lightweight systems.
Several lensless holographic and tomographic microscope approaches exist [8, 9, 13-26], with differing advantages and disadvantages. For example, use of incoherent light provides speckle noise reduction [15], but also reduced fringe visibility and depth of field. Tomography [17-20] can provide high resolution, but multiple reads are needed to acquire a full information set. Conversely, motion tracking does not necessarily require high-resolution imaging [8,21]. On-chip systems can provide a large field of view (FOV) by situating the sample very close to the detector array [21-23], but thermal issues can arise from differing ambient-sample and powered-detector temperatures. A more classical lensless DHM configuration with a somewhat larger sample-to-detector distance may thus be more suited to microscopic imaging in extreme environments. Of these, laser-based systems [14, 24-26] have tended to include pinholes and additional fore-optics to increase the laser-beam numerical aperture (NA), thereby increasing system volume and alignment complexity. For robustness and compactness, ideally any difficult-to-align elements such as small pinholes should be avoided, and any high-NA laser beam (or beams) should be provided without greatly impacting system volume. One or more embodiments of the present invention provide a solution to both of these issues by making use of small radial gradient-index (GRIN) rod lenses [27] or fibers to inject high NA laser beams into a lensless DHM.